(1) Field of the Invention
This application is concerned with certain peptide fragments of the p-17 gag protein of human immunodeficiency virus (HIV), to compositions containing the peptide fragment and its use for therapeutic, including vaccine, and diagnostic purposes, to DNA coding for the peptide fragment, and to methods for producing and using the peptide fragments and DNA encoding the fragments.
More particularly, this invention relates to peptide fragments having from about 30 to about 50 amino acids and which correspond to the Consensus C lade (subtype) of the p-17 gag protein, namely, extending over the region inclusive of, depending on the particular strain, amino acids at positions 75 to 129, and DNA fragments encoding this region.
(2) Discussion of the Prior Art
There has been extensive research over the past several years, first to identify the cause of AIDS and, after the positive identification of the retroviruses generically referred to as HIV, as the causative organism, efforts have concentrated on more detailed analysis of the genetic makeup, molecular biology, pathogenesis, biochemistry, development of highly sensitive methods of detection of virus and antibodies and treatments, and therapies. Extensive progress has been made in all of these areas yet much work needs to be done to effectively combat the spread of AIDS. While the very recent development of combination therapies utilizing protease inhibitors has had substantial success as a therapeutic treatment it is still very expensive and not universally effective. Therefore, still an essential part of the approach for combatting the spread of the highly infectious HIV virus and treating the disease in already infected individuals is the development of effective vaccines and immunotherapeutic agents that stimulate the appropriate components of the immune system. In this regard, knowledge of natural history of infection with HIV and the various immune responses and the clinical condition and the outcome may be useful in preparing effective vaccine preparations for either therapy or prevention. In this regard accurate diagnosis of the stage of disease and the immune status with regard to HIV and the knowledge that certain modalities are available may encourage infected patients to alter or modify their lifestyles in such manner as to reduce the risk of spreading the virus. Additionally, identification of protective antibodies based on epitope recognition can offer a more effective mechanism for staging and/or diagnosing the AIDS or pre-AIDS disease. Such staging and early diagnosis of seropositive individuals may then allow for vaccinations to provide the appropriate protective immune responses for treating the seropositive individual.
In commonly assigned U.S. Pat. No. 4,983,387 to A. Goldstein and S. Wang, the patentees describe an antigenic peptide extending from at least about position 92 to about position 109 of p17 gag protein of HIV-1. More specifically, however, what is described in this patent is a triacontapeptide of the formula:
(SEQ ID NO:3) Tyr Ser Val His Gln Arg Ile Asp Val Lys Asp Thr Lys Glu Ala Leu Glu Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Lys Ala.
(SEQ ID NO:3).
This triacontapeptide, referred to hereinafter as HGP-30, has shown positive results as a therapeutic agent against HIV, the causative organism of Acquired Immunodeficiency Deficiency Syndrome (AIDS) in certain clinical trials and in in vivo HIV challenge studies in SCID mice. One of the advantages of HGP-30, and the p-17 peptide, in general, is that this HIV protein tends to be more highly conserved across subtypes and strains of HIV than, for example, the envelope proteins. In fact, antibodies raised against HGP-30 in vaccinated individuals have been found by the present inventors to be recognized when tested against peptide sequences representing strains other than those represented by the HGP-30 sequence.
An interesting finding for HGP-30 is that this sequence contains both T and B cell epitopes immunoreactive with p17 of HIV (Sarin, et al., 1986, Science 232:1135; Naylor, et al., 1987, Proc. Nat. Acad. Sci. 84: 2951; Brander, et al., 1995, Clin. Exp. Immunol. 101:107-113; Dibrino, et al., 1994, J. Immunol. Vol. 154(2): 620-31). HGP-30 peptide has been conjugated to a large protein, Keyhole Limpet Hemocyanin (KLH), and found to be immunogenic in various animals and man, and the conjugate is well tolerated in both animals and humans (Gazzard, et al., 1992, Vaccine Res. 1:129; Sarin, et al., 1994, Vaccine Res. 3: 495; Kahn, et al., 1992, AIDS Res. Hu. Retroviruses 8:1321; Naylor, et al., 1991, Int. J. Immunopharm 13(Suppl): 117). A pilot study of HGP-30 vaccine has shown protection from HIV infection in such SCID Hu mice given PBMCs from an HGP-30 immunized donor (Sarin, et al., 1995, Cell. Molec. Biol. 41:401). More recently it has been shown that the presence of a predominance of IgG3 antibodies reactive to HGP-30 in sera of immunized human donors correlates with protection by PBMCs in the SCID Hu mouse HIV virus challenge model (Kahn et al 1996, Abstract 13 International AIDS Conference, Vancouver, Canada, July 1996; Talmadge, et al., 1996, Clin Immunol, Meeting New Orleans LA June 1996).
In particular, the sequence of HGP-30 is based on the HIV strain SF-2 which is a member of the B consensus sequence.
Prior work has established that p17 can be subdivided into several peptides for induction of immune responses (Goldstein, et al., U.S. Pat. No. 4,983,387; EP 0 246 829 Sarin, et al.; Jiang, et al., 1992, J. AIDS 5:382). That and other work has shown that in the p17 molecule numerous immunological epitopes are present for activities, such as B cell epitopes for induction of antibody, and T cell epitopes for helper activity and generation of cytotoxic T cells (Naylor, et al., 1990, Mono. Virol. 18:74; Coates, et al., 1987, Nature 326:549; Wahren, et al., 1989, J AIDS 4:448; Broliden, et al., ibid, Clin. Exp. Immunol. 76:216; Papsidero, et al., 1989, J. Virol 63:267; Boucher, et al., 1990, Clin. Lab. Anal. 4:43). Other work has shown that certain conjugates which include a T cell binding ligand (TCBL) and an epitope of interest from a disease associated antigen can also have biological activity even where the epitope alone is inactive (Zimmerman, et al., 1996a,b Vaccine Res. 5:91, 5:103; WO 89/12548).
It is difficult to predict whether changing the nature of a peptide, such as addition(s) or deletion(s) or substitution(s) of one or several amino acids, as occurs in different subtypes, could or would influence the subclass of antibody generated that recognize the epitope, even though it is known that such manipulations can induce different responses such as the stimulation of B and T cells, cytotoxic and lymphoproliferation responses. However, see our aforementioned U.S. Pat. No. 6,093,400, in which we demonstrated that a modified HGP-30 peptide, wherein the initial amino acid residue, corresponding to position 85, is shifted by at least 3 amino acid residues towards the N-terminal, i.e., beginning at the amino acid residue in the range of position 82 to 75 of the p17 gag peptide of HIV-1, induces a TH1 response. Nor is there evidence that the antibodies raised against a peptide fragment from one particular strain or subtype would exhibit different immunogenicity as compared to antibodies raised against corresponding peptide regions of other strains or subtypes.
It has now, surprisingly, been discovered, and this forms the basis of the present invention, that the peptide corresponding to HGP-30, but derived from Consensus C clade, elicits an even stronger and broad immune response than HGP-30 and, particularly, antibodies raised against a Consensus C sequence are able to recognize and give higher titers to HIV peptides from different subtypes and strains than antibodies to HGP-30.
Accordingly, it is an object of the invention to provide a novel peptide useful as a diagnostic aid for assaying for the HIV organism and/or as a prophylactic and therapeutic agent for treating AIDS or suppressing the formation of AIDS and having a T-cell epitope and, preferably also a B-cell epitope, and which has the ability to be recognized by antibodies raised against different subtypes and strains of HIV.